Multiplex Immunoassay for Serological Diagnosis of Mycobacterium bovis Infection in Cattle

Enfer Scientific, Unit T, M7 Business Park, Newhall, Naas, County Kildare, Ireland.
Clinical and vaccine Immunology: CVI (Impact Factor: 2.47). 11/2008; 15(12):1834-8. DOI: 10.1128/CVI.00238-08
Source: PubMed
ABSTRACT
Efforts to develop a better diagnostic assay for bovine tuberculosis have shown that the sensitivity and specificity of an assay can be improved by the use of two or more antigens. As reported here, we developed a multiplex chemiluminescent immunoassay that can simultaneously detect antibody activity to 25 antigens in a single well in a 96-well plate array format. The chemiluminescent signal is captured with a digital imaging system and analyzed with a macro program that tracks each serum for its pattern of antibody activity for Mycobacterium bovis antigens. The comparison of sera from 522 infected and 1,489 uninfected animals showed that a sensitivity of 93.1% and a specificity of 98.4% can be achieved with a combination of antigens. The assay system is rapid and can be automated for use in a centralized laboratory.

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CLINICAL AND VACCINE IMMUNOLOGY, Dec. 2008, p. 1834–1838 Vol. 15, No. 12
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Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Multiplex Immunoassay for Serological Diagnosis of Mycobacterium bovis
Infection in Cattle
Clare Whelan,
1
Eduard Shuralev,
1
Grainne O’Keeffe,
1
Paula Hyland,
1
Hang Fai Kwok,
2
Philip Snoddy,
2
Amanda O’Brien,
1
Marie Connolly,
1
Padraig Quinn,
1
Matt Groll,
3
Todd Watterson,
3
Sara Call,
3
Kevin Kenny,
4
Anthony Duignan,
5
Mary Jo Hamilton,
6
Bryce M. Buddle,
7
James A. Johnston,
2
William C. Davis,
6
Shane A. Olwill,
2
and John Clarke
1
*
Enfer Scientific, Unit T, M7 Business Park, Newhall, Naas, County Kildare, Ireland
1
; Fusion Antibodies Ltd., Unit 4,
Springbank Industrial Estate, Pembroke Loop Rd., BT17 0QL, Northern Ireland
2
; Quansys Biosciences, 365 North 600 West,
Logan, Utah 84321
3
; Central Veterinary Research Laboratory, Backweston, County Dublin, Ireland
4
; Department of
Agriculture and Food, Kildare St., Dublin 2, Ireland
5
; Department of Veterinary Microbiology and Pathology,
College of Veterinary Medicine, Washington State University, Pullman, Washington 99164-7040
6
; and
AgResearch, Hopkirk Research Institute, Palmerston North, New Zealand
7
Received 27 June 2008/Returned for modification 18 July 2008/Accepted 4 October 2008
Efforts to develop a better diagnostic assay for bovine tuberculosis have shown that the sensitivity and
specificity of an assay can be improved by the use of two or more antigens. As reported here, we developed a
multiplex chemiluminescent immunoassay that can simultaneously detect antibody activity to 25 antigens in a
single well in a 96-well plate array format. The chemiluminescent signal is captured with a digital imaging
system and analyzed with a macro program that tracks each serum for its pattern of antibody activity for
Mycobacterium bovis antigens. The comparison of sera from 522 infected and 1,489 uninfected animals showed
that a sensitivity of 93.1% and a specificity of 98.4% can be achieved with a combination of antigens. The assay
system is rapid and can be automated for use in a centralized laboratory.
Bovine tuberculosis (bTB) continues to be a zoonotic dis-
ease affecting multiple species, including humans (3, 9, 21, 26).
The disease has been difficult to control in livestock because of
the lack of an effective vaccine, the presence of wildlife reser-
voirs, and the lack of a diagnostic assay with sufficient sensi-
tivity and specificity to detect animals at all stages of infection.
Currently, the primary methods used for the detection of TB in
humans and ruminants include the measurement of a delayed-
type hypersensitivity (skin test) to purified protein derivative
(PPD) and an indirect in vitro assay that measures the con-
centration of gamma interferon (IFN-) produced in response
to stimulation with PPD (22, 30, 31). Although the methods
have proven useful in controlling bTB, they lack sensitivity and
specificity because of a cross-reactive immune response to T-
and B-cell epitopes conserved on orthologous molecules
present in nonpathogenic mycobacteria and Mycobacterium
avium subsp. paratuberculosis (reviewed in references 8, 23,
and 27). To obviate this problem, an extensive effort has been
under way to identify and characterize antigens unique to My-
cobacterium bovis that could be used in a diagnostic assay. To
date, studies have shown that the antibody response to M. bovis
is not uniform, with no evidence of a dominant persistent
response to a single antigen (reviewed in references 4, 7, and 8)
at any stage of infection (2, 19). This finding has suggested that
some type of a multiplex assay is needed to detect animals at
different stages of infection (1, 2). However, the necessity of
using multiple antigens in an assay has introduced another
challenge. The evaluation of the standard type of enzyme-
linked immunosorbent assay (ELISA) has shown that sensitiv-
ity and specificity are reduced when multiple antigens are com-
bined for analysis in a single well, thus limiting the way a
conventional ELISA can be used (20). To address this prob-
lem, we developed a multiplex assay that can simultaneously
detect and analyze the response to multiple antigens spotted in
a single well in a 96-well plate array format. We demonstrate
the enhanced diagnostic power of a multiplex antigen ap-
proach over that of the industry-standard methods (8).
MATERIALS AND METHODS
Serum samples. Serum samples used in the study were obtained from several
sources. Blood samples were taken into serum tubes (serum clot activator tubes;
Vacuette; Greiner-Bio-One), transported at room temperature, and then stored
at 2 to 8°C until processed. Following centrifugation (3,000 g for 30 min at 2
to 8°C) the serum was removed, aliquoted, and stored at 20°C.
The TB-negative sera were obtained from the Irish Department of Agriculture
from herds of animals with a known history of being free of M. bovis for at least
5 years. The TB-positive group of sera was collected from animals that were
proven to be positive for M. bovis infection at the time of slaughter based on
subsequent histopathological/bacteriological examination.
The third set of serum samples was from a bovine tuberculosis infectivity trial
undertaken by AgResearch (New Zealand). The sera were from 8-month-old calves
that were nonvaccinated but challenged via the intratracheal route with a low dose
of a virulent strain of M. bovis (approximately 5,000 CFU). Sera were collected prior
to challenge and then at 2, 5, 10, and 17 weeks postinfection (p.i.). A single intrad-
ermal comparative cervical tuberculin test (SICCT) was carried out prior to chal-
lenge and also during week 15 p.i. All animals in the study had lesions typical of a TB
infection consisting of a series of small lung lesions (diameter, 1 to 5 mm) or
pulmonary lymph node lesions ranging from 5 to 40 mm in diameter, and all animals
at 17 weeks p.i. were culture positive for M. bovis (Table 1).
Histopathology. Tissue sections were stained with hematoxylin and eosin for
histopathological examination. A diagnosis of tuberculosis was based on the
presence of a granulomatous lymphadenitis associated with areas of caseous
necrosis (6).
* Corresponding author. Mailing address: Enfer Scientific, Unit T, M7
Business Park, Newhall, Naas, County Kildare, Ireland. Phone: (353) 45
983800. Fax: (353) 45 983801. E-mail: johnclarke@enfergroup.com.
Published ahead of print on 15 October 2008.
1834
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Culture. The decontamination of tissues with 5% oxalic acid was performed as
described by Costello et al. (6). Samples were cultured on a MGIT 960 liquid
culture system (Becton Dickinson, MD) and Stonebrinks medium. Isolate iden-
tification was performed by AccuProbe (Gen-Probe Inc., San Diego, CA) and
GenoType MTBC (Hain Diagnostika, Nehren, Germany).
Preparation of antigens. The genes encoding different TB proteins were ex-
pressed in Escherichia coli as N-terminal polyhistidine-tagged (6 His) fusion
proteins by Fusion Antibodies Ltd. (Belfast) using the patented fusion expression
technology (FET) platform. Recombinant proteins were purified and polished to
near homogeneity by using the Fusion Antibodies Ltd. three-step chromato-
graphic protocol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
Western blot analysis were performed on all purified and polished recombinant
TB proteins to confirm that their level of purity was greater than 99%. Synthetic
peptides were synthesized by Genosphere Biotechnologies (France) to a purity
of 80% (by reverse-phase high-performance liquid chromatography at 220
nm). Amino acid residues were added to peptides to enhance the hydrophilicity
of the peptides. Lyophilized peptides were reconstituted to 2 mg/ml in sterile
phosphate-buffered saline, pH 7.4 (Sigma Aldrich, Dublin), and aliquots were
prepared and stored at 20°C. Antigen quantification was performed using the
micro-bicinchoninic acid protein assay kit (Pierce, Rockford, IL). See Table 2 for
the list of antigens (recombinant proteins and synthetic peptides) used in this
study.
Multiplex antigen printing. Antigens used in this study were printed in each
well of a black polystyrene 96-well plate in a multiplex planar array format by
Quansys Biosciences (Logan, UT). The optimization of the antigen printing was
carried out in conjunction with Enfer Scientific (Naas, Ireland). Plates were
printed by Quansys Biosciences, and the testing of plates was carried out at Enfer
Scientific. Antigen-coating concentration optimization also was carried out at
Enfer Scientific using a series of coating titrations printed in the optimized
printing buffer. In order to optimize the spot morphology, a fluorescent marker
was incorporated into all printing buffers at a concentration of 0.033 g/ml. This
allowed for visualizations of the spots using transillumination. Panels of antigens
were printed using the optimized printing protocol for sample testing. All plates
were shipped and stored at 2 to 8°C. Plates were allowed to warm to room
temperature for 30 min prior to use.
Multiplex immunoassay. The multiplex immunoassay method was developed
in-house at Enfer Scientific. Serum samples were diluted 1:250 into sample
dilution buffer (Enfer buffer A; Enfer Scientific) and mixed. A 30-l sample
dilution was added to each well. Sample dilution and plating were carried out
using an automated pipettor Tecan Genesis RSP 150 (Tecan, United Kingdom).
The plates were incubated at room temperature with agitation (900 rpm) for 30
min. The plates were washed with 1 Enfer wash buffer (Enfer Scientific) six
times and aspirated. The detection antibody (polyclonal rabbit anti-bovine im-
munoglobulin horseradish peroxidase; Dako, Denmark) was prepared to a dilu-
tion of 1:3,000 in detection antibody dilution buffer (Enfer buffer B; Enfer
Scientific). After the addition of 30 l of the detection antibody to test wells, the
plates were incubated at room temperature for 15 min with agitation (900 rpm).
The plates were washed as described above, and 30 l of substrate (50:50 dilution
of substrate and diluent; Quansys Biosciences, Logan, UT) was added per well.
Signals were captured during a 45-s exposure using a Quansys Biosciences Im-
aging system. Images were saved as CR2 image files. Data were extracted from
the captured images as relative light units (RLU) using the Quansys Q-View
software, version 2.0.
Anigen lateral-flow testing of sera. The testing of sera was carried out accord-
ing to the manufacturer;s instructions using the Anigen Rapid Bovine TB Ab
lateral-flow test kit (Animal Genetics Inc., Kyonggi-do, Korea). Following warm-
ing to room temperature, 4 drops of the sera were added to the sample depo-
sition area on the lateral-flow cassette. The test was read after 20 min. Only tests
with a positive control line were considered for further evaluation. In total, 214
TB-positive sera samples and 79 TB-negative samples were tested.
Data analysis. The data extracted from plate images with the Quansys soft-
ware were compiled and analyzed with a custom-made macro in Microsoft Excel
(Enfer multiplex macro, version 1.0.1.0). The macro was developed for the
multifactorial comparative analysis of the sensitivity and specificity of the mul-
tiple antigens used in a single well in a 96-well format. The analysis combines the
data from individual antigens and, based on a threshold for each, determines the
result of the test for any serum sample. A serum sample must be positive for a
minimum of any two antigens in a test well to be considered positive. This
method of analysis avoids the assessment of the sensitivity and specificity of
individual antigens used in the study and increases the overall accuracy of the
assay.
Statistical analysis was done on the infectivity study animal sample sets with
the Student’s t test using SigmaStat software (Systat, San Jose, CA).
RESULTS
Development of the multiplex immunoassay. Preliminary
studies were conducted to optimize and obtain the uniform
binding of each antigen using a fluorescent tag. Plates were
tested and evaluated by Enfer Scientific for signal variation
using a panel of serum samples. The testing of the plates with
the detection antibody, with and without sera from a panel of
control sera from uninfected cattle, showed that background
noise in the system in areas of the surface not coated with
antigens was, on average, 779.3 RLU. The optimization of the
printing concentration for all antigens was carried out on a
series of plates printed using sera from the TB-negative and
TB-positive samples. Optimal printing concentrations were de-
TABLE 2. Antigens used in this study
a
Protein
M. bovis
locus tag
-Crystalin_2...............................................................................Mb2057
HspX ............................................................................................Mb2057c
CFP-10.........................................................................................Mb3904
RPFc.............................................................................................Mb1916c
Rv2878 .........................................................................................Mb2904c
ESAT-6 ........................................................................................Mb3905
PPE68 ..........................................................................................Mb3903
Rv3616c .......................................................................................Mb3646c
Rv3879c .......................................................................................Mb3909c
MPB70 .........................................................................................Mb2900
MPB83 .........................................................................................Mb2898
Rv0283 .........................................................................................Mb0291
Rv2626 .........................................................................................Mb2659c
Rv1926c ........................................................................................Mb1961c
EsxH.............................................................................................Mb0296
PE35.............................................................................................Mb3902
Rv1573c .......................................................................................Mb1599
Rv1585c ........................................................................................Mb1611c
Rv1580c .......................................................................................Mb1606c
Rv1572c .......................................................................................Mb1598A
a
Antigens in boldface were investigated during this study but were not in-
cluded in the final panel of antigens.
TABLE 1. Summary of infectivity study samples
a
Sample
code
Lung lesion
score
b
Maximum diam
(mm) of lymph node
lesion
c
Tissues confirmed M. bovis
positive by culture
AM LB PM RB
A 5 15 15 Lungs; AM, LB, and PM
lymph nodes
B 0 12 5 8 8 AM, LB, PM, and RB
lymph nodes
C 3 8 Lungs; AM and LB lymph
nodes
D 5 15 40 30 Lungs; AM, LB, PM, and
RB lymph nodes
E 5 5 Lungs; AM, LB, PM, and
RB lymph nodes
a
All animals were confirmed to be without infection prior to challenge by
SICCT, and lesions were measured at 17 weeks p.i. Abbreviations for lymph
node locations: AM, anterior mediastinal; LB, left bronchial; PM, posterior
mediastinal; and RB, right bronchial.
b
Lung lesion scores: 0, no lesions; 1, 1 to 9 lesions; 2, 10 to 29 lesions; 2, 30 to
99 lesions; 4, 100 to 199 lesions; and 5, 200 lesions.
c
—, no lymph node lesion.
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termined for all antigens and then were tested with larger sets
of positive and negative sera before the subsequent testing of
all samples for the determination of overall assay sensitivity
and specificity. Assay optimization was carried out based on
initial work done at Enfer Scientific using a single-plex ELISA
on Nunc Maxisorp 96-well microtiter plates. The optimal ex-
posure time was determined to be 45 s.
The bioinformatic analysis of pathogenic and environmental
strains of mycobacteria facilitated the identification of a series
of proteins that were likely to be antigenic (i.e., cell surface or
secreted) and disease selective. In total, we report on 20 anti-
gens and highlight the most important 13 that can be used in
the diagnosis of M. bovis infection in cattle (Table 2).
A total of 1,489 TB-negative and 522 TB-positive sera were
screened against the panel of antigens. The diagnostic value of
each antigen was calculated based on its ability to correctly
identify a known positive/negative sample. This allowed us to
select individual thresholds (cutoffs) for each antigen that best
suited their inclusion in a multiplex format. In certain in-
stances, the sensitivity of an antigen was sacrificed by increas-
ing the threshold in order to improve its specificity. The indi-
vidual sensitivities of the antigens ranged from 5.4 to 95.0%,
while individual specificities ranged from 69.1 to 99.1% (Table
3). The in-house macro was used to run a combinatorial data
analysis to calculate the highest specificity and sensitivity based
on the panel of antigens with the cohorts of samples tested. A
two-positive-protein rule was applied to the analysis. For a
sample to be considered a true positive, a response to at least
two antigens must be observed above the individual thresholds
for each of the antigens being tested. This rule increases the
overall accuracy of the assay. The individual specificities and
sensitivities for three antigens currently under study (ESAT-6,
CFP-10, and MPB83) are compared to that of the multiplex
assay and a commercial antigen assay in Table 3.
Screening of infectivity study samples. A total of five ani-
mals that were challenged with a dose of approximately 5,000
CFU M. bovis were tested on the multiplex assay. Serum sam-
ples were tested from the animals at the following time points:
prechallenge (time zero) and then 2, 5, 10, and 17 weeks p.i.
All animals were positive by the 10th week p.i. using the mul-
tiplex assay. Three of the five animals were detected at 5 weeks
p.i. (Table 4). The time points at which each animal was de-
termined to be positive on the Enfer multiplex immunoassay,
as well as results for ESAT-6, CFP-10, and MPB83 antigens,
are shown (Table 4). Figure 1A shows a representative histo-
gram in terms of the signal (in RLU) obtained from test wells
at the indicated time points for one of the infected animals
(animal A), which showed a positive response by week 5. A
representative image for a panel of antigens is shown in
Fig. 1B.
Comparison of tests. To compare the sensitivity and speci-
ficity of the multiplex assay to those of an assay based on a
single antigen, we conducted a set of experiments with a com-
mercial assay available to us, the Anigen lateral-flow assay. The
Anigen lateral-flow test kit uses a recombinant MPB70 antigen
for the detection of antibodies. The results with the sera se-
lected for the comparison with the Enfer multiplex immuno-
assay are shown in Table 3. The analysis of results from the
Enfer multiplex immunoassay showed a specificity of 98.4%
and a sensitivity of 93.1% (1,489 negative and 522 positive
sera). The analysis of the Anigen lateral-flow kit showed a
specificity of 84.2% and a sensitivity of 84.0% (79 negative and
214 positive sera) (Table 3).
DISCUSSION
Cumulative studies have emphasized that the development
of a robust rapid diagnostic assay for M. bovis infection will
require the use of a multiplex system that can simultaneously
TABLE 3. Cohorts of samples tested by Enfer multiplex immunoassay
and the Anigen lateral-flow kit and individual responses to
ESAT-6, CFP-10, and MPB83 antigens
Test
TB-positive
animals
(no.)
Sensitivity
(%)
TB-negative
animals
(no.)
Specificity
(%)
Enfer multiplex
immunoassay
522 93.1 1,489 98.4
ESAT-6 522 40.6
a
1,489 86.6
a
CFP-10 522 82.6
a
1,489 69.7
a
MPB83 522 78.5
a
1,489 99.1
a
Anigen lateral
flow
214 83.6
b
79 83.0
c
a
Data were extracted from the multiplex assay for these individual antigens.
b
The sensitivity of the Anigen lateral-flow kit is 90.0% compared to that of
culture-confirmed positives, and it is 85.1% compared to that of skin test-con-
firmed positives according to the manufacturer’s instructions.
c
The specificity of the Anigen lateral-flow kit is 98.6% compared to that of
skin test-confirmed negative animals according to the manufacturer’s instruc-
tions.
TABLE 4. Results for animal serum samples from the infectivity
study prechallenge and at 2, 5, 10, and 17 weeks postchallenge
Sample
code
Wk
Assay result
Enfer
multiplex
immunoassay
ESAT-6
a
CFP-10
a
MPB83
a
A0 ⫺⫺
2 ⫺⫺
5 ⫹⫺
10 ⫹⫺
17 ⫹⫺
B0 ⫺⫺
2 ⫹⫺
5 ⫺⫺
10 ⫹⫺
17 ⫹⫺
C0 ⫺⫺
2 ⫺⫺
5 ⫺⫺
10 ⫹⫹
17 ⫹⫹
D0 ⫺⫺
2 ⫺⫺
5 ⫹⫺
10 ⫹⫺
17 ⫹⫺
E0 ⫺⫺
2 ⫺⫺
5 ⫹⫺
10 ⫹⫺
17 ⫹⫺
a
Data were extracted from the Enfer multiplex immunoassay for these indi-
vidual antigens.
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detect and analyze antibody activity in response to multiple
antigens in a serum sample (1, 2, 7, 19, 29). The various ap-
proaches taken to develop such an assay have included the
examination of latex bead agglutination, immunochromato-
graphic assays (2, 12, 13, 20), fluorescence polarization (15,
28), electrochemiluminescence (17, 32), and chemilumines-
cence (14). As reported here, the chemiluminescence-based
multiplex platform developed by Quansys Biosciences for use
in nonveterinary assays has proven to be an ideal platform for
the development of a multiplex assay for bTB (14, 25). The
initial use of the assay with a panel of expressed antigens and
peptides, including some of the best-known antibody targets in
M. bovis infection, has shown that the overall sensitivity and
specificity is increased by using more than one antigen in the
assay. A specificity of 98.4% and a sensitivity of 93.1% were
obtained with a cohort of known M. bovis-positive and -nega-
tive sera using a minimum criterion of a positive reaction with
any two antigens in the array. Other groups also recognized a
higher level of diagnostic accuracy through the use of multiple
antigens. Multiantigen print immunoassay lateral-flow devel-
opments (18, 20) and the use of fusion proteins (16) both
illustrate the potential for increasing sensitivity by employing
combinations of suitable antigens.
The five-animal infectivity study reported here, using the
multiplex assay, demonstrates that the assay has the capability
of identifying infection as early as 2 weeks p.i. Furthermore, all
animals were successfully identified as positive by 10 weeks p.i.
Further infectivity study trials are planned that will utilize
serial samples from animals receiving different challenge doses
and will be assessed for reactivity with panels of antigens on
the multiplex platform.
Currently, the available tests (using SICCT and IFN-) have
been reported to cover a range of sensitivities and specificities
(6), and the combined use of these tests has the potential to
increase the overall sensitivity and specificity of the testing.
With these tests come some restrictions; for example, routine
SICCTs can be carried out only at 60-day intervals (EU Di-
rective 64/432, European Economic Community). The IFN-
assay is a blood-based assay that may be influenced by the time
lapse between blood sampling and conducting the assay, as
reported by Gormley et al. (10, 11).
The application of the multiplex assay alone or in combina-
tion with the current SICCT and IFN- methods (6, 10, 11) of
identifying M. bovis-infected animals could improve the detec-
tion of infected animals at earlier stages of infection. In addi-
tion, the improved ability to screen multiple M. bovis antigens
on a versatile multiplex platform provides an opportunity to
develop a better diagnostic test not only for cattle but also for
other species. Species that could be considered in this context
are other domestic ruminants and potential bTB wildlife res-
ervoirs, such as deer and badgers. Recent reports emphasize
that there is also a need for the development of a suitable ante
mortem bTB test for these species (5, 24).
ACKNOWLEDGMENTS
We thank all of the staff who participated in the project at Enfer
Scientific; AgResearch staff who contributed to the infectivity study;
Eamonn Costello and the staff of the Tuberculosis Laboratory Central
Veterinary Research Laboratory (Backweston, Co. Kildare), where the
culture and histopathology work was carried out; and Margaret Good
and the staff of the Irish Department of Agriculture and Food, Dublin
2, Ireland, for sample acquisition.
This study was funded by Enfer Scientific.
FIG. 1. (A) Data (in RLU) from the multiplex immunoassay obtained for animal A in the infectivity study group of animals showing
representative results. Note that the pattern of response varied for each individual animal tested (*MPB70 is a peptide preparation). Significance
(P 0.05) between the prechallenge sample and the postchallenge samples are indicated by an asterisk. (B) Representative image for a panel of
antigens. Each well shown represents a sample point (prechallenge and 2, 5, 10, and 17 weeks p.i.) for one animal.
V
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1838 WHELAN ET AL. CLIN.VACCINE IMMUNOL.
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    • "PCR methods have also been described. New diagnostic blood tests include lymphocyte proliferation assay, IFN-γ assay and enzyme-linked immunosorbent assays (ELISA) [11,12] . IFN-γ assay is only useful in members of bovidae however lymphyocyte proliferation test may be used on other animals and wild animals. "
    Full-text · Article · Jan 2015 · Kafkas Üniversitesi Veteriner Fakültesi Dergisi
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    • "The strips are incubated with serum samples and then is followed by immunodetection using standard chromogenic methods (Lyashchenko et al., 2000Lyashchenko et al., , 2008 Veerasami et al., 2012). Other researchers have developed and assayed a multiplex chemiluminescent immunoassay that can simultaneously detect antibody directed to more than 20 antigens in a single well in a 96- well plate format that has been commercialised as Enferplex TB (Whelan et al., 2008Whelan et al., , 2010bWhelan et al., , 2011). Based on previous studies, the Se and Sp yielded using both kind of assays was not significantly different from that obtained when using tests based on a single antigen (Table 2). "
    [Show abstract] [Hide abstract] ABSTRACT: Bovine tuberculosis (TB), mainly caused by Mycobacterium bovis, is a zoonotic disease with implications for Public Health and having an economic impact due to decreased production and limitations to the trade. Bovine TB is subjected to official eradication campaigns mainly based on a test and slaughter policy using diagnostic assays based on the cell-mediated immune response as the intradermal tuberculin test and the gamma-interferon (IFN-γ) assay. Moreover, several diagnostic assays based on the detection of specific antibodies (Abs) have been developed in the last few years with the aim of complementing the current diagnostic techniques in the near future. This review provides an overview of the current ante-mortem diagnostic tools for diagnosis of bovine TB regarding historical background, methodologies and sensitivity (Se) and specificity (Sp) obtained in previous studies under different epidemiological situations.
    Full-text · Article · Oct 2014 · Research in Veterinary Science
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    • "tested MPB83 and MPB70 using sera from infected cattle and reported apparent sensitivity and specificity of 63% and 98%, respectively [16]. ESAT-6 and CFP-10 were used in an ELISA format by Whelan et al., 2008 using a total of 1489 bovine TB negative and 522 bovine TB positive cattle sera and stated sensitivity of 40.6% - 86.6% and specificity of 82.6% -69.7%, based upon use of two different test interpretation criteria [26]. In the present study, we detected anti-M. "
    [Show abstract] [Hide abstract] ABSTRACT: Background The use of serological assays for diagnosis of bovine tuberculosis (TB) has been intensively studied and use of specific antigens have aided in improving the diagnostic accuracy of the assays. In the present study, we report an in-house enzyme linked immunosorbent assay (ELISA), developed by using ethanol extract of Mycobacterium bovis (M. bovis). The assay, named (ethanol vortex ELISA [EVELISA]), was evaluated for detection of anti- M. bovis antibodies in the sera of cattle and white-tailed deer. Methods By using the EVELISA, we tested sera obtained from two species of animals; cattle (n = 62 [uninfected, n = 40; naturally infected, n = 22]) and white-tailed deer (n = 41 [uninfected, n = 25; naturally infected, n = 7; experimentally infected, n = 9]). To detect species specific molecules, components in the ethanol extract were analyzed by thin layer chromatography and western blotting. Results Among the tested animals, 77.2% of infected cattle and 87.5% of infected deer tested positive for anti- M. bovis antibody. There were only minor false positive reactions (7.5% in cattle and 0% in deer) in uninfected animals. M. bovis -specific lipids and protein (MPB83) in the ethanol extract were detected by thin layer chromatography and western blotting, respectively. Conclusion The results warrant further evaluation and validation of EVELISA for bovine TB diagnosis of traditional and alternative livestock as well as for free-ranging animal species.
    Full-text · Article · Jul 2014 · BMC Veterinary Research
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